The hypothesis to be tested in this U19 program is that combining gene modifying reagents with different modes of action will have a significant impact on HIV-1 disease with the possibility of achieving a cure. We will build upon our previous extensive experience in anti-HIV-1 genetic therapies to both broaden our knowledge and develop new technologies that will result in lentiviral vector based anti-HIV-1 therapeutic development candidate(s). The plan is to develop 2 vectors, one for HSPC and one for T-cell transplant and file an IND for a Phase I clinical trial by the end of the grant term. The few gene-based therapies for HIV-1 disease that have been tested in the clinic have been focused on protecting the differentiated progeny T-cells and macrophages, principally through ablation or reduction of CCR5 expression. In the single remarkable case of the Berlin patient, allogeneic transplant of CCR5?32 donor cells resulted in a functional cure without evidence for remaining HIV-1. Efforts to mimic this CCR5 ablation through transplant of gene-engineered cells has shown some success, but suffers from several roadblocks which we will address in this proposal. First, a universal limitation in stem cell transplant is the difficulty of achieving engraftment levels sufficient to provide therapeutic efficacy. We propose to address this fundamental issue by testing approaches to selectively enrich for repopulation of gene-modified hematopoietic stem/progenitor cells (HSPC) using genetic selection for engrafted cells. A second major issue, one faced by all HIV-1 therapies, is the development of resistance by HIV-1. As with the development of small molecule therapies for HIV-1 disease, gene therapies will also require effective combinations. As such, our corporate partner, Calimmune, Inc., is currently testing in humans, T-cell and HSPC genetic therapy using CCR5 knockdown (shRNA1005) combined with a transmembrane fusion inhibitor (C46). Here, we propose to add a third reagent, a chimeric antigen receptor (CAR) recognizing HIV-1 infected cells, delivered by adoptive T-cell immunotherapy. T-cell immunotherapy with tumor specific CARs has proven to be effective against cancer in early human studies. While a CAR was tested years ago in humans for HIV-1 disease and found to be safe, it suffered from a number of limitations, now better understood, and to be addressed here. Finally, HSPC and T-cell transplants are complex biological processes that require a thorough understanding of repopulation by thousands of functionally diverse stem, progenitor, or mature cells. Each of the project leaders has had extensive experience working not only with HIV-1, but also in general stem cell biology and its applications to HIV-1 disease. The breadth of expertise ranges from vector and transgene development (Chen, An, Kitchen, Symonds), development and use of animal models for HSPC biology (Kitchen, An, Chen), anti-HIV-1 immune function (Yang, Kitchen), understanding of HSPC behavior (Chen) to Phase I and II clinical trial implementation (Symonds).